Ethylene is a well-established plant hormone. It plays an important role in virtually every phase of plant development including seed germination, fruit ripening, leaf and flower senescence, and abscission. The production of ethylene may also be induced by external factors such as mechanical wounding, anaerobiosis, auxin treatment, ultraviolet light, temperature extremes, water stress, and ions such as cadmium, and lithium ions (Abeles, F. B., 1973, Ethylene in Plant Biology, 197-219, Academic Press, London; Yang & Hoffman, 1984, Annu. Rev. Plant Physiol., 35, 155-189).
The pathway for ethylene biosynthesis has been established, the first step of which involves the formation of S-adenosyl-L-methionine (AdoMet) by S-adenosyl-L-methionine synthetase. AdoMet is subsequently converted by S-adenosyl-L-methionine methylthio-adenosine-lyase (ACC synthase; EC 4.4.1.14) to the nonprotein amino acid 1-aminocyclopropane-1 carboxylic acid (ACC), the immediate precursor of ethylene in higher plants (Adams & Yang, 1979, Proc. Natl. Acad. Sci. U.S.A., 76, 170-174). Physiological analysis has suggested that this is the key regulatory step in the pathway, (Kende, 1989, Plant Physiol., 91, 1-4). Thus, the rate of endogenous expression of ACC synthase is considered to limit substantially the rate of ethylene production.
It appears that ACC synthase is encoded by a highly divergent multigene family (for a review, see Theologis, A. 1992, Cell, 70, 181-184; Kende, H., 1993, Annu. Rev. Plant Physiol. Plant Mol. Biol., 44, 283-307). In tomato, for example, ACC synthase is encoded by at least six genes, two of which are expressed in fruit ripening (Van der Straeten et al., 1990, Proc. Natl. Acad. Sci. U.S.A., 87, 4859-4863; Olson et al., 1991, Proc. Natl. Acad. Sci. U.S.A., 88, 5340-5344; Rottmann et al., 1991, J. Mol. Biol., 222, 937-961; Yipp et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89, 2475-2479).
In addition, reference may be made to an article by Theologis, A. (1992, supra) in which the structure and expression of 20 ACC synthase genes were compared from a variety of plant species including winter squash, zucchini, tomato, Arabidopsis, apple, rice, mung bean and carnation. This comparison suggested that the extensive polymorphism and distinct regulatory networks governing the expression of ACC synthase subfamilies arose early in plant evolution, prior to the divergence of monocotyledons and dicotyledons.
It is well known that endogenous ethylene is often deleterious to crops. In particular, increased ethylene production due to trauma caused by mechanical wounding of fruits and vegetables, and the cutting of flowers greatly diminishes their post harvest quality and storage life. Thus, it has been a major goal of postharvest physiologists to effect suppression of fruit ripening and flower fading by inhibiting the biosynthesis of ethylene.
To this end, a strategy has been developed that takes advantage of the modulation properties of ACC synthase in the control of ethylene biosynthesis. In this regard, reference may be made to International Application Publication No. WO 92/04456 which is directed to inhibition of expression of endogenous ACC synthase using an antisense expression system. This system comprises a DNA molecule capable of generating, when contained in a plant host cell, a complementary RNA that is sufficiently complementary to an RNA transcribed from an endogenous ACC synthase gene to prevent the synthesis of endogenous ACC synthase. Ethylene production in fruits of transgenic tomato plants engineered using this system was inhibited by 99.5% and, as a consequence, fruit ripening was suppressed. In addition, the application of ethylene or propylene to the fruits of these plants restored normal ripening.
Thus, ACC synthase genes may be used as targets for the generation of transgenic plants in which endogenous expression of ACC synthase is inhibited to effect suppression of ethylene production and a concomitant delay in fruit ripening.
The efficacy of this system, however, is predicated on the condition that the antisense RNA is sufficiently complementary to the transcript expressed from the target gene. Accordingly, if there is diversity between different ACC synthase genes, the use in this system for example, of a particular ACC synthase gene from one plant species would not be expected to inhibit the expression of an ACC synthase gene from another plant species. This is supported on page 5 of WO 92/04456 which states: "While the various ACC synthases are generally active in a variety of plant tissues, the DNAs are not completely homologous, and therefore the use of the genetic materials for control of synthesis, for example, using an antisense strategy, does not translate cross species."